JP2002209574A - Gene disrupted yeast - Google Patents

Abstract

(57) [Summary] [Problem] To use yeast characteristics and to construct an industrially useful substance production system by genetic recombination, a yeast in which only a specific gene is disrupted to add auxotrophy is used. provide. SOLUTION: By homologous recombination with chromosomal DNA using an ADE1 gene fragment encoding Candida maltosa phosphoribosylaminoimidazole-succinocarboxamide synthase (EC 6.3.2.6),
A yeast strain in which the ADE1 gene has been disrupted is prepared. Further, the disrupted strain is transformed with a gene expression cassette containing a biodegradable polyester synthase gene, and the transformant is cultured to produce a biodegradable polyester.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for disrupting a specific chromosomal DNA of yeast by the principle of homologous recombination, and a disrupted strain. In addition, the present invention relates to the production of industrially useful substances using the disrupted strain.

[0002]

2. Description of the Related Art The development of genetic recombination technology has made it possible to produce industrially useful substances in large quantities using prokaryotes and eukaryotes. Among the eukaryotes, yeast, Saccharomyces, has been used for the production of fermentable foods such as alcoholic beverages for a long time, and Candida maltosa (C
andida maltosa) was once used as a microbial protein in foods and feeds, and the safety of yeast itself has been confirmed.

[0003] Yeast grows fast and can generally be cultured at higher cell densities than bacteria. In addition, yeast is easier to separate bacterial cells and culture solution than bacteria, and thus can simplify the process of extracting and purifying products. Utilizing these characteristics, yeast can be used for recombinant DN.
A is used as a production host of a useful product by A,
Its usefulness has been demonstrated.

[0004] Among various yeasts, Candida yeast differs from Saccharomyces in that it does not produce ethanol in aerobic culture and is not inhibited from growing by ethanol. Thus, efficient cell production and substance production are possible. Furthermore, the spore-free yeast Candida maltosa has the property of being able to assimilate and grow using straight-chain hydrocarbons having a carbon chain of C _{6 to} C ₄₀ and oils such as palm oil and coconut oil as sole carbon sources. . Since this property is practically advantageous as a production site of a useful substance or a reaction field by converting a hydrophobic chemical substance, it is expected to be used for production of various compounds (Reference book: Non-Conventional, edited by Wolf K.). Yeasts i
n Biotechnology. A Handboo
k, Springer-Verlag, Berlin
(1996) p411-580). It is also expected that Candida maltosa can be used for producing useful substances by genetic recombination utilizing its properties, and gene expression systems for this purpose have been vigorously developed (Japanese Patent Application Laid-Open No. Sho 62). -74287, JP-A-62-74288, JP-A-2-72822). Recently, it has been published that Candida maltosa can be used for the production of linear dicarboxylic acids by genetic recombination (WO
99/04014).

[0005]

When yeast is used as a host for substance production by genetic recombination, it is necessary to obtain a mutant strain to which a selection code such as an appropriate auxotrophy is added, as in the case of using Escherichia coli or the like. There is. Conventionally, to add auxotrophy, mutants have been obtained by random mutagenesis using a mutagen such as nitrosoguanidine or ethyl methanesulfonic acid. However, although the target auxotroph can be obtained by this mutagenesis method, the possibility that the mutation is present in a portion other than the target site cannot be denied. This is an obstacle to the development using yeast as a host, and it can be said that the utilization as a place for producing substances is delayed as compared with E. coli and the like.

[0006] For example, a host vector system for Candida maltosa was developed from an early stage, and despite the fact that a large number of auxotrophic mutants have been obtained by mutagenesis, recombinants have been obtained. The production of new useful chemical substances used has not been industrialized. The reason for this is that no Candida maltosa having appropriate auxotrophy with the same growth ability as that of the wild type and the ability to assimilate linear hydrocarbon chains has not been obtained. C developed by mutating Candida maltosa
It has been confirmed that the HA1 strain has mutations in the ADE1 gene and the HIS5 gene (Kawai S. et al.
gric. Biol. Chem. , 55: 59-65.
(1991)), the growth ability is inferior to the wild type. This is thought to be due to mutations other than the target site.

[0007] Another problem of the mutant strain obtained by random mutation is the spontaneous reversion of the mutation site. In this case, since the revertant strain grows preferentially during the culture, the productivity of the recombinant bacterium may decrease. In addition, the reverted strain is more likely to survive and proliferate when leaked to the natural world, and there is a problem in terms of safety standards. Therefore,
It is not appropriate to use a strain obtained by random mutagenesis as a place for producing a substance. Therefore, it has been desired to obtain a disrupted strain in which only a specific auxotrophic gene is disrupted.

[0008] As described above, mutants of Candida maltosa include ADE1 gene and histidinol-phosphate-aminotransferase (HIS5).
Gene), orotidine-5'-phosphate decarboxylace (URA3 gene), etc. (Reference book: Wolf K. Ed.
conventional Yeasts in Bi
technology. p411-580). However, Candida maltosa that has added auxotrophy by disrupting only certain genes has not been obtained to date. In order to utilize the characteristics of yeast and to construct an industrially useful substance production system by genetic recombination,
Such a gene-disrupted strain has been desired. Further, not only Candida maltosa, but also such a gene-disrupted strain is desired.

[0009] In addition, Candida maltosa is expected to produce industrially useful substances utilizing the property of utilizing and growing straight-chain hydrocarbons, palm oil, palm oil and other fats and oils as the sole carbon source. It had been.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by utilizing gene recombination techniques, it was possible to synthesize yeast phosphoribosylaminoimidazole-succinocarboxamide in yeast. Enzyme (EC
The ADE1 gene-disrupted strain was produced based on the principle of homologous recombination with chromosomal DNA using a DNA (ADE1 gene) fragment encoding 6.3.2.6), and an adenine-requiring yeast was successfully obtained. . Then, the growth ability and gene expression ability of the gene-disrupted yeast were determined by the mutation treatment of the ADE1 gene mutant strain of Candida maltosa.
The present invention was completed by showing that both abilities were superior to that of the CHA1 strain as compared with A1.

That is, the present invention relates to a yeast in which the ADE1 gene of chromosomal DNA has been disrupted by homologous recombination with an ADE1 DNA fragment. The present invention also relates to a transformant of an ADE1 gene disrupted yeast transformed with a DNA sequence containing a homologous or heterologous gene.

More specifically, Candida
By providing a transformant in which a heterologous gene expression system has been introduced into an ADE1 gene-disrupted strain of maltosa, a method for producing an industrially useful substance is provided. Specifically, a two-component copolymerized polyester of 3-hydroxybutyrate (hereinafter abbreviated as 3HB) and 3-hydroxyhexanoate (hereinafter abbreviated as 3HH) useful as a biodegradable polyester ( Hereinafter, P (3HB-co
-3HH)), a polyester synthase gene and an enoyl-CoA hydratase gene expression system.
DE1 gene-disrupted strain, and thereby P (3HB
-Co-3HH) and the ADE
(1) The usefulness of the gene disruption Candida maltosa was demonstrated. That is, the present invention also relates to a method for producing a polyester using a transformant of the ADE1 gene-disrupted strain, wherein the polyester is collected from a culture obtained by culturing the transformant. Hereinafter, the present invention will be described in detail.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION There is no particular limitation on the yeast which disrupts the ADE1 gene by homologous recombination, and the genus Acicloconidium deposited in a strain depository (eg, IFO, ATCC, etc.).
Genus), Ambrosiozyma
genus ma), Arthroascus (Arthroasc)
genus), Arxiozyma (Arxiozyma)
Genus), genus Ashbya (genus Ashbya), genus Babjevia (genus Babjevia), genus Bensingtonia (genus Bensingtonia), genus Botryaoscus (genus Botryaoscus), genus Botryozyma (genus Botryozymat), genus Bottazymaes, o Burella (
Genus Bullella), genus Bulleromyces (Bull)
eromyces), Candida (Candid)
genus a), genus Citeromyces
Genus), Clavispora (Clavispora)
Genus), Cryptococcus
genus), Cystophilus genus (Cystof)
genus irobasidium), genus Debaryomyces (genus Debaryomyces), genus Deccala (D
genus ekkara), genus Dipodascopsis (Dipo)
genus dascopsis), Dipodascus genus (Dip
odacus), Eeniella (Eeniella)
Genus), Endomycops
ella), Eremascus (Eremascus)
Genus), the genus Eremothecium
Genus), Erythrobasidium (Erythroba)
genus sidium), pheromyces (Felom)
yces), Filobasidium (Filoba)
genus sidium), galactomyces (Galac)
genus tomyces), Geotricum (Geotri)
genus chum), genus Guillermondella (Guillie)
rmondella), Hansenia spora (Ha)
genus nseniaspora), Hansenula (Han)
genus senula), Hasegawa (Hasegawa)
aea), Holtermannia (Holterma)
genus nnia), Hormoascus (Hormoas)
genus cus) and the genus Hyphopich
ia), Isatchenkia (Issatchen)
Kia), Kloeckera (Kloeckera)
Genus), Chloekeraspora (Kloeckersasp)
ora), Kluyveromyces (Kluyver)
genus omyces), genus Kondoa (genus Kondoa),
Genus Kuraisia (genus Kuraisia), genus Kurtzmanomyces (genus Kurtzmanomyces), genus Leukosporidium (Leucosporidiu)
m), the genus Lipomyces (genus Lipomyces), the genus Roderomyces (genus Roderomyces), the genus Malassezia (genus Malassezia), the genus Metshnikowia (genus Metschnikowia), the genus Murakia (genus Mrakia), and the genus Moxozai
genus yxozyma), genus Nadsonia (Nadsoni)
genus a), genus Nakazawaae (Nakazawaea)
Genus), Nematospora (Nematospora)
Genus), genus Ogataea (genus Ogataea), genus Ausporidium (genus Oosporidium), genus Patissolen (genus Pachysolen), genus Faticospora (genus Phachtychospora), genus Phaffia (genus Phaffia), genus Pichia (genus Pichia)
a), Rhodosporium (Rhodospor)
genium, genus Rhodotorula
genus la), Saccharomyces
ces), Saccharomyces (Saccha)
genus romycodes), Saccharomycopsis (genus Saccharomycopsis), genus Cytoella (genus Saitoella), genus Sakagutia (Sa
genus kaguchia), genus Satanospora (Satu
genus rnospora), schizoblast sporion (
(Schizoblastosporion genus), Schizosaccharomyces (Schizosaccharom)
genus yces), Schwaniomyces (Schwan)
niomyces), Spolidiobolas (S
genus poridiobolus, genus Sporobolomyces, genus Sporopachydermia, genus Stefanoascus
Genus), genus Sterigmatyses (Sterigmat)
omyces), Sterigmatosporidium (
Genus Steigmatosporidium), genus Symbiotaphrina (genus Symbiotaphrina),
Sympodiomyces genus
genus), Sympodiomycopsis (Sympodi)
omycopsis), Torulaspora (Toru)
laspora) and Trichosporia (Tric)
hosporiella), Trichosporon (T
richosporon), Trigonopsis (T
rigonopsis), Tsuchiyaea (Tsuc)
genus hiyaea), Udeniomyces (Udeni)
omyces), Walt Myces (Waltom)
yces), Wikerhamia (Wickerha)
genus Mia), Wikerhamiera (Wickerha)
genus miella), Williopsis (Williop)
genus sis), Yamadazyma genus
Genus), genus Yarrowia (genus Yarrowia), genus Zygoascus (genus Zygoascus), genus Zygosaccharomyces (Zygosaccharomyces)
Genus), Zygowilli (Zygowilli)
opsis genus or Zygozym genus
Yeast such as genus a) can be used.

Among these yeasts, the genus Candida, the genus Yarrowia, the genus Kluyveromyces, and the genus Hansenula are preferable because they enable efficient cell production and substance production by continuous culture at high density. In particular, the genus Candida is preferred in that host-vector systems have been studied and the system can be easily used, and the ability to assimilate linear hydrocarbons and oils and fats is high.

As an example of the production of the ADE1-disrupted strain of the present invention and its use, Candida maltosa was used.

The wild type strain CAM12247 of Candida maltosa used in the present invention can be obtained from the Institute of Applied Microbiology of the University of Tokyo or American
anType Culture Collection
(Manassas, VA, USA). The CHA1 strain, which is a mutant of Candida maltosa adenine and histidine auxotrophic marker, is A
It is available from the above organization as TCC90625.

The ADE1 gene-disrupted strain Candida maltosa AC16 strain obtained in the present invention has been internationally deposited under the deposit number of FERM BP-7366 at the Patented Microorganisms Depositary Center in the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry. I have.

[0018] Homologous recombination refers to recombination occurring at a portion where the base sequence of DNA is similar or has the same sequence (homologous sequence).

[0019] Gene disruption is the act of mutating the base sequence of a gene, inserting another DNA, or deleting a portion of the gene so that the function of the gene cannot be exerted. Is shown. As a result of gene disruption, the gene cannot be transcribed into mRNA, and the structural gene is not translated or transcribed mRN.
Since A is incomplete, the amino acid sequence of the translated structural protein is mutated or deleted, so that the original function cannot be exhibited.

The ADE1 gene is a 5 'untranslated region including a promoter region and a phosphoribosylaminoimidazole-succinocarboxamide synthase (EC 6.3.
The figure shows a gene fragment consisting of a region encoding 2.6) and a 3 ′ untranslated region including a terminator region. The nucleotide sequence of the ADE1 gene of Candida maltosa is Ge
nBank: D00855 and the DNA sequence is shown in SEQ ID NO: 1.

The ADE1 DNA fragment is a DNA capable of causing homologous recombination with the ADE1 gene on the chromosome in a microbial cell, thereby destroying the ADE1 gene.
A is shown.

The ADE1 enzyme is phosphoribosylaminoimidazole-succinocarboxamide synthase (EC
6.3.2.6).

The homologous gene means a gene existing on the chromosome of the host yeast or a partial DNA thereof.
A heterologous gene means a gene that is not originally present on the chromosome of the host yeast or a partial DNA thereof.

[0024] The gene expression cassette includes a transcription promoter DNA sequence and a DNA encoding a gene to be expressed.
DNA containing NA and terminator to terminate transcription
And those that function extrachromosomally in the form of circular plasmids, and those that integrate into chromosomal DNA.

The gene expression product is itself a gene expression product when the substance expressed by the gene (gene expression product) is a desired protein or enzyme. Gene expression products are various enzymes and coenzymes, and substances which are produced by the enzymes expressing catalytic activity in host yeast and are directly different from gene expression products are also gene expression products.

PHA is an abbreviation of polyhydroxyalkanoate, and represents a biodegradable polyester obtained by copolymerizing 3-hydroxyalkanoic acid.

ORF2 refers to a DNA designed to express a gene encoding a polyester synthase derived from Aeromonas caviae (JP-A-10-108682) in Candida maltosa. SEQ ID NO: 6 shows the nucleotide sequence.

ORF3 refers to a DNA designed to express a gene encoding enoyl-CoA hydratase derived from Aeromonas caviae (Japanese Patent Laid-Open No. 10-108682) in Candida maltosa.
SEQ ID NO: 7 shows the nucleotide sequence.

Hereinafter, an example of a method for producing an ADE1 gene-disrupted strain and a method for producing a polyester using the disrupted strain will be described.

(1) Method for Producing ADE1 Gene-Disrupted Strain For producing a gene-disrupted strain, any method can be used as long as a disrupted strain that does not express the ADE1 enzyme is obtained. Although various methods for gene disruption have been reported, gene disruption by homologous recombination is preferred in that only a specific gene can be disrupted (Nickol
off J.C. A. Edition Methodsin Molec
ural Biology, 47: 291-302 (1
995), Humana Press Inc. , T
otowa, NJ)). Among homologous recombination, a disrupted strain that does not return spontaneously can be obtained, and as a result, a strain that is highly safe in handling the recombinant can be obtained.
Step-destruction method (one-step gene disru)
ption) is preferred.

As the ADE1 DNA fragment used in the one-step disruption method, usually, a DNA fragment in which a partial DNA inside a gene is removed and the remaining both ends are ligated again is used.
This DNA fragment can be prepared, for example, by PCR (polymerase chain reaction) or by excision and religation from a vector with a restriction enzyme. ADE
The length of the homology region at both ends of one DNA fragment may be 10 bases or more, preferably 200 bases or more, and more preferably 300 bases or more. Further, the homology of each end is preferably 90% or more, more preferably 95% or more.

The partial DNA to be removed is ADE by removal.
One gene is a portion where the enzyme activity cannot be exhibited and the ADE1 enzyme activity is not recovered by spontaneous reversion. The chain length of such a partial DNA is not particularly limited, but is preferably 50 bases or more, more preferably 1 base or more.
It is at least 00 bases.

In the present invention, about 553 bp of the DNA fragment encoding the ADE1 enzyme is removed, and the 5'-side 630 bp
Disruption DNA in which an NA fragment and a 3′-side 400 bp DNA fragment were ligated was used (FIG. 1). Removed part is ADE1
It accounts for about 80% of the enzyme protein. In addition, 5 'side and 3'
The homology between the DNA fragment on the side and the original ADE1 gene is
NA is 100%. DNA for destruction used in the present invention
The nucleotide sequence of the fragment is shown in SEQ ID NO: 2.

The preparation of the DNA fragment used in the present invention is carried out using pU
C119-ADE1 (Kawai S. et al., Agric.
Biol. Chem. 55: 59-65 (199).
1)). That is, plasmid pUC1
Escherichia coli transformed with 19-ADE1 is cultured, and a plasmid is prepared therefrom. This plasmid is cut with an appropriate restriction enzyme so as to remove a part of the structural gene of the ADE1 gene (gene encoding the ADE1 enzyme protein), and the vector is recovered and ligated again using ligase. Thus, a vector containing the DNA for disruption is obtained.

The vector containing the DNA for disruption is introduced into an appropriate E. coli, for example, JM109 or DH5α, and the E. coli is cultured in a large amount, and then a high-purity plasmid is prepared in a large amount by ultracentrifugation with cesium chloride (Sambrook et al. Molecular cloning: A Lab
laboratory Manual, Second Edi
Tion 1.42-1.47, Cold Spring
g Harbor Laboratory Press
(1989)). This vector can be used directly for gene disruption, but in order to perform the one-step disruption method, a homologous portion containing the ADE1 region is cut out from the purified vector with an appropriate restriction enzyme, and is used as DNA for disruption. It is desirable. In the present invention, the ADE1 gene could be disrupted by homologous recombination by cutting with a restriction enzyme SalI and introducing the DNA fragment into the cells without purification.

The Candida maltosa transformation method includes a protoplast method and a lithium acetate method (Takagi method).
M. Et al., J Bacteriol, 167: 551-.
5 (1986)), the electric pulse method (Kaskeke)
A. Yeast 8: 691-697 (199
2)) is known, but in the present invention, the electric pulse method was used. A commercially available device can be used for electric pulse generation. In the present invention, BTX (San Diego, CA)
USA) ELECTRO CELL MANIP
ULATOR 600 was used. The cuvette is BIO
MEDICAL CORPORATION CO. LT
D (Tokyo Japan) BM6200 (2m
m gap blue cap) was used. After the electric pulse, the cells are cultured in an appropriate medium, and a target ADE1-disrupted strain is screened from the obtained cultured cells.

Various methods for obtaining transformants have been developed, of which nystatin enrichment (Snow)
R. Nature 211: 206-207 (196
6)) can be used. This method is a method developed for efficiently selecting mutant strains obtained by random mutation from yeast, but is considered to be applicable to gene-disrupted strains. For example, according to a preferred embodiment of the present invention, after the electric pulse, the cultured cells are inoculated into a minimal medium or the like and cultured. The bacteria are washed and cultured in a minimal medium containing no nitrogen source, and then cultured for a short time in a minimal medium containing a nitrogen source. Nystatin was added directly to the culture and added for 1 hour 3
By aerobically culturing at 0 ° C., wild strains can be preferentially killed. This bacterial solution is spread on an appropriate agar medium plate, and cultured at 30 ° C. for about 2 days to obtain a red colony.

From the obtained red colonies, PCR or genomic Southern hybridization (Sambr) was used.
book, etc., Molecular cloning: A
Laboratory Manual, Second
Edition 9.31-9.57, Cold S
spring Harbor LaboratoryPr
ess (1989)), gene disruption can be easily confirmed. In the PCR method, when both ends of the ADE1 gene are used as primers, a normal size DNA band is detected in a wild type strain in agarose gel electrophoresis, but a band shorter by the deleted sequence is detected in a disrupted strain. Is done. In the present invention, a DNA band of about 1 kbp was detected. However, in the case of replacement or integration with chromosomal DNA such as gene disruption, it is necessary to assume that the gene may be inserted into a part other than the intended one, for example, into an unknown part having high homology. You may not be able to confirm. The PCR method is desirably used as a means for selecting a candidate strain when a large number of disrupted strains are obtained.

Genomic southern analysis is indispensable for confirming a disrupted strain or the like. By carrying out this method, it can be easily and surely proved that the gene recombination has occurred only at the intended site. Chromosomal DNAs of the disrupted strain and a wild-type strain as a control are prepared, cut with an appropriate restriction enzyme, and then subjected to agarose gel electrophoresis. In the present invention,
Confirmation was attempted by three kinds of cutting methods using two kinds of restriction enzymes. Transfer the DNA from the agarose gel onto a nylon membrane and use the ADE that is considered to remain on the genome of the disrupted strain.
HpaINdeI digested DNA fragment 2 of one gene
70 bp was enzymatically labeled using a GeneImage Labeling / Detection Kit (Amersham) according to the attached instructions to obtain a probe for Southern hybridization detection. After hybridization, the plate was washed, and a DNA band was detected with a fluorescent coloring reagent according to the attached instructions. The detection band was compared with that of the wild strain (IAM12247). As a result, it was confirmed that 550 bp inside the ADE1 gene was shortened by the three cutting methods for the two strains.

The obtained ADE1 gene-disrupted strain was ADE
(1) Adenine, which is a precursor of ATP, cannot be synthesized because the enzyme cannot be synthesized. Therefore, it cannot survive in a medium without adenine or in an environment without adenine.

One of the obtained ADE1 gene disrupted strains was named Candida maltosa AC16. (2) Expression of heterologous gene by ADE1 gene-disrupted strain: method for producing polyester A homologous gene or a heterologous gene can be expressed using the ADE1-disrupted strain obtained in the present invention. Since yeast can secrete a glycosylated protein, which cannot be produced by Escherichia coli, into a medium, such a protein can be produced using an ADE1-disrupted strain.

The homologous gene that can be introduced is not particularly limited, and for example, as an example of producing an industrially useful product, WO
Production of dicarboxylic acids by introducing a P450 enzyme gene derived from the same strain into Candida maltosa, as disclosed in 99/04014. Further, the heterologous gene is not particularly limited, and examples thereof include production of the protein by introduction of a lipase gene, an amylase gene, and the like.

In some Candida yeasts such as Candida maltosa used in the present invention, some codons are translated differently from other organisms at the stage of protein translation from mRNA. It is known. In Candida maltosa, the leucine codon CUG is translated into serine (Ohama T. et al., Nucl.
eic Acid Res. , 21: 40394045.
(1993)), the lacZ gene derived from Escherichia coli is not translated into β-galactosidase having activity (Sugiy)
ama H .; Et al., Yeast 11: 43-52 (19
95)). When such a heterologous gene is expressed, there is no guarantee that it will be translated into a functional protein in Candida maltosa. Therefore, when expressing a heterologous gene using Candida maltosa as a host, only leucine codon may be converted in principle, but other amino acid codons may be matched to those of Candida maltosa for more efficient expression.
Codon conversion is described, for example, in Wolf K. et al. Hen Nonco
eventual Years in Biot
technology. Mauersberger in
S. Et al., Candida maltosa p52
4-527 may be used as a reference.

As the gene involved in polyester synthesis, any gene may be used as long as it is a gene involved in the synthesis of polyester represented by the following general formula. The following general formula shows the structural formula of polyester P (3HB-co-3HH), wherein X and Y are integers, and 3HB and 3HH, respectively.
Shows the number of polymerizations.

[0045]

Embedded image For example, a polyester synthase gene fragment described in JP-A-10-108682 can be used. Further, a gene involved in polyester synthesis may be introduced together with the present polyester synthase gene. These genes include, for example, enoyl-CoA, an intermediate in the β-oxidation pathway, with (R) -3-hydroxyacyl-C
(R) -body-specific enoyl-CoA hydratase that converts to oA (Fukui T. et al., FEMS Microb
ology Letters, 170: 69-75.
(1999), JP-A-10-108682),
Β-ketothiolase / NADH-dependent hydratase gene that synthesizes 3-hydroxybutyryl-CoA by dimerizing acetyl-CoA (Peoples OP, J.B.
iol. Chem. 264: 15298-15303
(1989)). As long as these genes have substantial enzymatic activity, mutations such as deletions, substitutions, and insertions may be made in the base sequences of the genes. However, in the case of a heterologous gene, there is no guarantee that it will be translated into a protein having a function in Candida maltosa as described above, and therefore, it is necessary to change the amino acid codon.

In this study, a polyester synthase derived from Aeromonas caviae and enoyl-CoA hydratase (JP-A-10-108682, Fukui T., et al.
FEMS Microbiology Lette
rs, 170: 69-75 (1999). However, since there is a high possibility that this enzyme gene is not normally translated and does not show enzymatic activity, the DNA was completely synthesized by matching the amino acid codons of both enzymes to the optimal codons of Candida maltosa. Total Synthesis of Polyester Synthase from Aeromonas caviae and Enoyl-CoA Hydratase DN
The A sequence is shown in SEQ ID NOs: 6 and 7 in the sequence listing, respectively.
However, the nucleotide sequences shown in these SEQ ID NOs are not limited thereto, and any nucleotide sequence may be used as long as the amino acid sequence of the enzyme is a nucleotide sequence expressed in Candida maltosa. it can.

The gene expression cassette in yeast is composed of a promoter connected to the 5 'upstream of the gene and a DNA sequence such as a 5' upstream activating sequence (UAS), a polyA addition signal and a terminator 3 'downstream of the gene. Etc. D
It is prepared by linking NA sequences. Any of these DNA sequences can be used as long as they function in the yeast.

There are promoters that constantly express and those that inducibly express. Although any promoter may be used, it is preferable that the promoter is derived from the same Candida maltosa as the host. As an example, the ALK1 promoter and ALK1 of Candida maltosa
Terminator (GenBank D00481) (T
akigi M .; Agric. Biol. Che
m. 5: 2217-2226 (1989)).

The gene expression cassette used for the transformation of the present invention can be constructed, for example, as follows. The vector used for the construction may be any vector as long as it is a plasmid that autonomously propagates in Escherichia coli,
Further, the yeast may have a region capable of autonomous growth. Vectors capable of autonomous propagation in yeast are known to be retained in cells. Also, the gene expression cassette can be integrated into the chromosome. As an example, p capable of autonomous propagation in Candida maltosa
UTU1 can be used (M. Ohkuma, e.
tal j. Biol. Chem. , Vol. 27
3, 3948-3953 (1998)).

In the present invention, the promoter ALK1p (SEQ ID NO: 8) and the terminator ALK1t (SEQ ID NO: 9) of the Alk1 gene of Candida maltosa were ligated 5 ′ upstream of ORF2 and ORF3, respectively.

To prepare a restriction enzyme site for linking a structural gene to a promoter and a terminator, a PCR method can be used. The primer sequences used for PCR are shown in SEQ ID NO: 10 to SEQ ID NO: 13. As PCR conditions, any conditions may be used as long as the target gene fragment can be amplified.

The promoter portion uses SEQ ID NO: 8 as a template, SEQ ID NO: 10 and SEQ ID NO: 11, and the 5 '
ALK1p having allI and the 3 ′ end at NdeI can be prepared. The terminator portion can be used to prepare ALK1t having HindIII at the 5 ′ end and EcoRV at the 3 ′ end, using SEQ ID NO: 9 as a template and SEQ ID NO: 12 and SEQ ID NO: 13. The vector pUTA1 in which the marker gene is changed from uracil to adenine using pUTU1 and the Ade1 gene of Candida maltosa can be used as the vector. pUCNT (WO
94/03613)), binds ALK1t to the PvuII and NdeI sites, and binds HindI to pUCNT.
II, ALK1t binds to SspI site and pUAL
1 can be constructed. Next, Nde of pUAL1
I and PstI sites were ligated with ORF2, and plasmid p
UAL-ORF2 can be constructed. Also, pU
PUCNT-ALK1 constructed during construction of AL1
By binding ORF3 to the NdeI and HindIII sites of t and further binding ALK1p, pUAL-O
RF3 can be constructed.

Next, an upstream promoter and a downstream terminator were cut out together with ORF2 from plasmid pUAL-ORF2 using EcoT22I and ligated to the PstI site of pUTA1.
A-ORF2 can be constructed. Furthermore, pUA
Using SalI from L-ORF3, together with ORF3, a promoter located upstream and a terminator located downstream are cut out together to construct a plasmid pUTA-ORF23 (top in FIG. 2) bound to the SalI site of pUTA-ORF2. By the above method, a gene expression cassette for producing polyester in Candida maltosa can be constructed.

The strain of the present invention was transformed with the gene expression cassette using the above-mentioned transformation method, and was transformed into pUTA-ORF.
Candida maltosa AC16 (OR
F23) strains can be prepared.

Production of polyester by culturing yeast transformed with a gene expression cassette involved in polyester synthesis can be performed as follows.
As the carbon source used for the culture, any carbon source can be used as long as yeast can assimilate. When the expression of the promoter is inducible, an inducer may be added as needed. The inducer may be the primary carbon source. As a nutrient other than the carbon source, a medium containing a nitrogen source, inorganic salts, and other organic nutrients can be used. The culture temperature may be any temperature at which the bacterium can grow, but is preferably 20 ° C to 40 ° C. The culture time is not particularly limited, but may be about 1 to 7 days. Thereafter, the polyester may be recovered from the obtained cultured cells or culture.

As the carbon source, glucose, glycerin,
Carbohydrates such as sucrose, oils and fats, fatty acids, and further n-alkanes can be used. Examples of fats and oils include rapeseed oil, coconut oil, palm oil, palm kernel oil and the like. Examples of the fatty acid include saturated and unsaturated fatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and myristic acid, and fatty acid derivatives such as esters and salts of these fatty acids. . As an example, in the cultivation of Candida maltosa, cultivation can be performed using oils and fats as a carbon source. In the case of fats and oils that cannot be assimilated or cannot be efficiently assimilated, it can be improved by adding lipase to the medium. Furthermore, by transforming the lipase gene, the ability to assimilate fats and oils can be imparted.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as peptone, meat extract, yeast extract and the like. Examples of the inorganic salts include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, and sodium chloride.

Other organic nutrients include amino acids,
Examples include glycine, alanine, serine, threonine, proline and the like, and vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, vitamin C and the like.

Many methods have been reported for the recovery of polyester from bacterial cells. In the present invention, for example, the following method can be used. After completion of the culture, the culture is separated and recovered from the culture with a centrifuge or the like, and the cells are washed with distilled water, methanol and the like, and then dried. The polyester is extracted from the dried cells using an organic solvent such as chloroform. Cellular components are removed from the organic solvent solution containing the polyester by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the polyester. The precipitated polyester is removed by filtration or centrifugation to remove the supernatant and dried to recover the polyester.

Analysis of the obtained polyester is, for example,
It can be performed by gas chromatography, nuclear magnetic resonance, or the like.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention does not limit the technical scope to these examples.

[0062]

EXAMPLES As reagents used for culturing yeast, those sold by Wako Pure Chemical Industries were used unless otherwise specified.

(Medium composition) LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L salt. For LB plate, add 16 agar
g / L. YPD medium: 10 g / L yeast extract, 20 g / L polypeptone, 20 g / L glucose. In the case of a YPD plate, agar is added to 20 g / L. In the case of an adenine-containing YPD medium, 0.1 g / L of adenine is added. YM medium: 3 g / L yeast extract, 3 g / L malt extract, 5 g / L bactopeptone, 10 g / L glucose. SD medium: 6.7 g / L amino acid-free yeast nitrogen base (YNB), 20 g / L glucose. In the case of an adenine-containing medium, 24 mg / L of adenine is added.
In the case of an SD plate, agar is added so as to be 20 g / L. M medium: 0.5 g / L magnesium sulfate 0.1 g / L salt, 0.4 mg / L thiamine, 0.4 mg / L pyridoxine, 0.4 mg / L calcium pantothenate, 2 mg
/ L inositol, 0.002 mg / L biotin, 0.
05 mg / L iron chloride, 0.07 mg / L zinc sulfate, 0.1 mg / L
01mg / L boric acid, 0.01mg copper sulfate, 0.01m
g potassium iodide, 87.5 mg / L potassium dihydrogen phosphate, 12.5 mg / L potassium dihydrogen phosphate, 0.1 g
1 g / L calcium chloride, 20 g / L glucose. In the case of M medium containing ammonium sulfate, 1 g / L ammonium sulfate is added. M containing ammonium sulfate and adenine
In the case of a medium, 1 g / L ammonium sulfate and 24 mg / L adenine are added to M medium. Medium for polyester production: 6.7 g / LYNB, 10 g
/ L casamino acid is added with 20 g / L of n-alkane or fat as a carbon source.

The liquid culture of yeast was performed in a 50 ml test tube, 50 ml.
This was performed using a 0 ml Sakaguchi flask or a 2 L Sakaguchi flask. 300 rpm, 500 m for 50 ml test tube
Shaking culture was performed at 100 to 110 rpm in the case of 1 Sakaguchi flask and 90 to 100 rpm in the case of 2 L Sakaguchi flask.
The culture temperature is 30 ° C. for both liquid culture and plate culture.

(Restriction Enzyme Treatment) Restriction enzyme treatment may be performed under the reaction conditions recommended by the manufacturer, or edited by Sambrook et al., Molecular cloning: A Lab.
laboratory Manual, Second Edi
, Cold Spring Harbor L
The method was performed according to the method described in Laboratory Press (1989).

In the practice of the present invention, a number of commercially available kits were used, but were carried out according to the attached instructions unless otherwise specified.

(Example 1) Preparation of DNA for disruption Vector pUC119- for Escherichia coli containing the ADE1 gene
ADE1 (Kawai S. et al., Agric. Biol.
Chem. , 55: 59-65 (1991)) (4.6).
4 kbp) was cut with MunI and HpaI, and then TAKAR
A smoothing process is performed using A Blunting Kit (Takara Shuzo). The divided DNA that has been subjected to the blunting treatment is 1
Then, the vector and ADE1 both sides (4.19 kbp) and the MunI / HpaI digestion fragment (550 bp) are separated. Both sides of the vector and ADE1 were recovered from agarose gel using EASYTRAP (Takara Shuzo), and TAKARA Ligati was collected.
ADE using on Kit Ver2 (Takara Shuzo)
One side was connected. The reaction solution was added to 100 μl of Escherichia coli competent cell DH5α (manufactured by Takara Shuzo) at a solution ratio of 5% or less to transform the Escherichia coli. LB
An appropriate amount of medium is added, and after shaking culture for 1 hour, smeared on an LB plate and cultured overnight at 37 ° C. Several emerging colonies were obtained, cultured in an LB medium by a conventional method, and plasmid DNA was extracted from the cultured cells. This plasmid is replaced with the restriction enzyme Sa
Cleavage with I1 and agarose gel electrophoresis 1.03 kb
The appearance of the p band was confirmed, and the target disruption vector was obtained. The transformed Escherichia coli was cultured in a large amount in an LB medium, and was subjected to Molecular cloning: A Lab.
laboratory Manual, Second Edi
Tion 1.42-1.47, Cold Spring
g Harbor Laboratory Press
According to (1989), the vector was prepared in a large amount by ultracentrifugation using cesium chloride. Then, in order to perform the direct disruption method, the ADE1 DNA for disruption was cut out with SalI.
An aqueous solution having a total DNA amount of 5 mg / ml was prepared, and the cut-out DNA was used for gene disruption without purification.

(Example 2) Gene disruption experiment Candida maltosa IAM12247 strain was inoculated into 10 ml of YM medium and pre-cultured overnight. Next, preculture 1
The resulting mixture was inoculated into 100 ml of YM medium (500 ml Sakaguchi flask), cultured for 6 hours, and collected by centrifugation. The bacteria are suspended in 500 μl of 1M sorbitol, and the DNA for disruption is suspended.
100 μg was added and the mixture was gently stirred.

Gene transfer was performed by the electric pulse method.
Gene transfer device is ELECTRO CEL manufactured by BTX
L MANIPULATOR 600 was used. The cuvette is BIO MEDICAL CORPORATION
N CO. BM6200 manufactured by LTD was used. 100 μl of the prepared competent cell / DNA solution was taken, injected into a cuvette, and set in a pulse device. Subsequently, an electric pulse was applied under the conditions of a capacitance of 40 μF, a resistance value of 246 ohm, and a voltage of 1.9 KV. After the pulse, 1 ml of 1M sorbitol was added to each cuvette, mixed gently, and left at room temperature for 1 hour. Then, YM medium 100m
1 and cultured overnight.

Example 3 Screening of Disrupted Strains Next, bacteria cultured in a YM medium were inoculated into 50 ml of an ammonium sulfate-free M medium (500 ml Sakaguchi flask) and cultured overnight. Subsequently, 100 m of M medium containing ammonium sulfate
and cultured at 30 ° C. for 6 hours. To this culture solution, a 3 mg / ml ethanol solution of nystatin (manufactured by Sigma) was added to a final concentration of 10 μg / ml, and the cells were cultured for 1 hour. Wash the nystatin-treated bacteria twice with sterile water,
Resuspended again in 50 ml of sterile water. This bacterial solution was spread on a YPD plate. When the plate was cultured at 30 ° C. for 2 days, 24 red colonies were obtained. It was confirmed that none of these strains could grow on SD medium.

(Example 4) Analysis by PCR method Primary screening of disrupted strains from a large number of red colonies was performed by PCR. The obtained red colony is YP
The cells were cultured in a medium D, and chromosomal DNA was extracted therefrom using Gentle-kun (Takara Shuzo), a chromosome extraction kit. Using this chromosomal DNA, PCR was performed using primers (SEQ ID NO: 3 and SEQ ID NO: 4) at both ends of the ADE1 gene. PCR is Perkin Elmer
This was performed using Amplitaq kit. Gene amplification was performed using Biometer's TRIO-Thermoblo.
ck ^TM was used. The cycle was performed at 94 ° C for 1 minute, 50 ° C for 1 minute, and 70 ° C for 3 minutes, and 30 cycles were performed. 1.5 kb of DNA is amplified in the wild strain, and 1. kb in the gene-disrupted strain.
DNA shortened to 0 kb is amplified. The PCR reaction solution was subjected to electrophoresis on a 1% agarose gel, and 1.0 kbp
Was detected. As a result, the ADE1 gene in 3 out of 24 strains was shortened and considered to be disrupted. FIG. 3 shows the PCR patterns of two out of three strains.

(Example 5) Analysis by Genomic Southern Hybridization Method The chromosomal DNA of these three strains was extracted using Gentle-kun (Takara Shuzo), a chromosome extraction kit. 5 μg of the obtained chromosomal DNA was used for each of three methods, DraI and Dr.
The cells were cut with a + NdeI and NdeI and electrophoresed on a 0.8% agarose gel. Molecular clon
ing: A Laboratory Manual, S
second edition 9.31-9.57, C
oldSpring Harbor Laborato
The gel was transferred to a Hybond N + filter (Amersham) overnight according to ry Press (1989). Southern blot detection probe is ADE
HpaIndeI fragment (270
bp) was used with an enzyme-labeled GeneImage labeling / detection kit (Amersham). After hybridization, wash and DN with the fluorescent reagent of the same kit.
A band was detected. The detection band was used for the wild strain IAM122.
When compared with that of the 47 strains, 550 bp within the ADE1 gene was shortened in 2 strains (FIG. 4), and disruption of the ADE1 gene on the chromosome was confirmed. FIG. 5 shows ADE1 as revealed by Southern hybridization.
Genetic Disruption A restriction map of the Candida maltosa chromosome near the disrupted ADE1 gene is shown. 553
bps indicates the portion deleted between MunI and HpaI. “probe” indicates a partial DNA used for Southern hybridization. As shown in FIG. 4, when this probe was used, the length of the base described below the chromosomal DNA was confirmed. In the wild strain IAM12247, a value obtained by adding 553 bps to this value was confirmed.

One of the ADE1 gene-disrupted strains thus obtained was named Candida maltosa AC16.

Example 6 Investigation of Proliferation Ability of ADE1 Gene-Disrupted Strain A complete medium of gene-disrupted AC16 strain (adenine (100 m
g / L) and histidine (100 mg / L)
Medium) was compared with IAM12247 strain and CHA1. The yeast was inoculated into 10 ml of YPD medium, cultured overnight, and then inoculated into 10 ml of the same medium at 1%. The OD 600 nm was measured at regular intervals. As a result, the AC16 strain and the wild strain reached the end of culture in 15 hours, but the AC16 strain grew only up to 83% of the wild strain. On the other hand, CHA1
Grew to a concentration equivalent to that of the AC16 strain, but the AC16 strain was 6 hours earlier (FIG. 6). Thus, AC16
It was shown that the strain had better growth ability in a complete medium than the CHA1 strain.

(Example 7) Examination of fat assimilation ability of ADE1 gene-disrupted strains Among the gene-disrupted strains, the fat and fat assimilation ability of AC16 strain was compared with that of wild-type strains IAM12247 and CHA1 strain. As fats and oils, palm oil, coconut oil, and tetradecane which is an n-alkane having 14 carbon atoms were used. YNB with 2% oil added
The cells were inoculated into 50 ml of a medium (500 ml Sakaguchi flask) and cultured overnight. This bacterial solution was inoculated into the same amount of medium at 10%, and the OD600 nm was measured at regular intervals. As a result, in palm oil and palm oil, the AC16 strain obtained this time is about 15% inferior in growth ability to the wild strain, but has about 4 times as much assimilation capacity as the CHA1 strain (6 times in 32 hours). I understand. In the case of tetradecane, CHA1 showed higher growth ability than fats and oils, but it was 55% of wild type and 80% of AC16 strain. Thus, the ADE1 disrupted strain is CHA
The ability to utilize fats and oils and alkanes was superior to one strain. FIG.
The growth curves for each carbon source are shown in FIG.

Example 8 Synthesis of Genes Involved in Polyester Synthesis As enzyme genes involved in polyester synthesis, PHA synthase derived from Aeromonas caviae and enoyl-CoA, an intermediate in the β-oxidation pathway, are monomers ( R)-
(R) form-specific enoyl-CoA hydratase which converts to 3-hydroxyacyl-CoA (T. Fukui, et.
al FEMS Microbiology Lett
ers, vol. 170, 69-75 (1999)), and designed the enzyme gene.

In designing the nucleotide sequence, Candida maltosa is a yeast that translates CTG codon into serine instead of leucine.
Did not assign. The codon corresponding to each amino acid was selected preferentially for the frequently used codons in Candida maltosa. Codon usage is Wo
If K. Hen Nonconventional Ye
asts in Biotechnology. In Mauersberger S. Et al., Candida
maltosa p524-527 was referred to. ORF2 and ORF3 designed in SEQ ID NOs: 6 and 7, respectively
showed that. DNA was totally synthesized according to the nucleotide sequence.

Example 9 Construction of Recombinant Plasmid for Synthesizing Polyester The ORF2 and ORF3 were placed 5 ′ upstream from each other so that the ORF2 and ORF3 were expressed in Candida maltosa.
Maltosa Alk1 gene promoter ALK1p
(SEQ ID NO: 8) was linked to the terminator ALK1t (SEQ ID NO: 9) of the Alk1 gene of Candida maltosa at the 3 ′ downstream. In order to prepare a restriction enzyme site for connecting a promoter and a terminator to a structural gene, a PCR method was used. The PCR conditions were 94 ° C. for 1 minute, 55 ° C. for 2 minutes, and 72 ° C. for 3 minutes, and this was repeated 25 times to amplify the target gene fragment. The polymerase used was ExTaq from Takara Shuzo Co., Ltd. The promoter portion uses SEQ ID NO: 8 as a template, SEQ ID NO: 10 and SEQ ID NO: 11, and the 5 ′ end is Sal.
ALK1p having I and 3 ′ ends at NdeI was prepared. The terminator part was prepared using SEQ ID NO: 9 as a template.
And SEQ ID NO: 13, the 5 ′ end was HindIII,
ALK1t at the 3 ′ end was EcoRV. Finally, the vector linking ORF2 and ORF3 contains pUC1
9 shows the self-replicating region of Candida maltosa (AR
S) (Gen Bank D29758) and URA
PUTU (M. Ohkuma, et al, J. B.) linked to three genes (GenBank D12720).
iol. Chem. , Vol. 273, 3948-
3953 (1998)) and pUTA1, a vector in which the marker gene was changed from uracil to adenine using the ADE1 gene of Candida maltosa (FIG. 2).
Bottom) was used. pUTA1 is derived from pUTU1 by Xho
The URA3 gene was removed using I, and the ADE1 gene fragment cut out using SalI was ligated to construct the URA3 gene.

ALK1p was bound to the PvuII and NdeI sites of pUCNT (described in WO94 / 03613), and ALK1t was bound to the HindIII and SspI sites of pUCNT to construct pUAL1. Then p
ORF2 was bound to the NdeI and PstI sites of UAL1 to construct a plasmid pUAL-ORF2. Also,
pUCNT-AL constructed during construction of pUAL1
By binding ORF3 to the NdeI and HindIII sites of K1t and further binding ALK1p, pUAL
-ORF3 was constructed.

Next, an upstream promoter and a downstream terminator were cut out together with ORF2 from the plasmid pUAL-ORF2 using EcoT22I, and ligated to the PstI site of pUTA1.
A-ORF2 was constructed. Furthermore, pUAL-ORF3
Using SalI together with ORF3, an upstream promoter and a downstream terminator were cut out together to construct a plasmid pUTA-ORF23 (top in FIG. 2) bound to the SalI site of pUTA-ORF2.
By the above method, a gene expression cassette for producing polyester in Candida maltosa was constructed.

Example 10 Preparation of Transformant for Polyester Production Transformation into the AC16 strain and the CHA1 strain was performed by culturing the yeast in a YM medium until the OD600 nm reached 0.8 to 1.0, followed by centrifugation. The cells were collected. The above expression vector was introduced into the cells. When transfecting, from preparation of competent cells to gene transfer, GENO TEC
Company H FastTrack ^™ -Yeast Trans
This was performed using the formation _SM Kit. The transformed cells were smeared on an M medium plate and cultured for 2 days. Since the ADE1 gene is incorporated in pUTA-ORF23, adenine can be synthesized. Therefore, a bacterium that grows on an adenine-free medium is a transformant. The bacteria grown from the adenine-free medium were collected and used as a transformant AC16 (ORF23). Similarly, the CHA1 (ORF23) strain was obtained from the CHA1 strain. Further, a control vector pUTA1 containing no polyester synthase was used.
Was used to transform the AC16 strain and the CHA1 strain in the same manner, and the transformants AC16 (CT) strain and CHA1 (O
(RFCT) strain was obtained.

Example 11 Polyester Production Culture All main cultures for polyester production were performed using a 2 L Sakaguchi flask. First, 100 μl of the glycerol stock of each recombinant strain was inoculated into 10 ml of SD medium and cultured overnight. 1 ml of the cultured yeast was inoculated to 50 ml of an ammonium sulfate-containing M medium (100 g / L ammonium sulfate added) for starvating glucose. After culturing in this medium for 2 days, glucose in the medium is completely depleted. Then, 10 ml of this bacterium was added to 50 ml of a polyester production medium (5 ml).
(00 ml Sakaguchi flask), and after culturing for 8 hours, 500 ml of the same polyester production medium was inoculated in its entirety and main culture was performed. The main culture was performed for 120 hours. After autoclaving the culture, the cells were collected by centrifugation, washed with methanol, and lyophilized. The obtained dried cells were pulverized, 100 ml of chloroform was added, and the mixture was stirred overnight to perform a polyester extraction operation. The chloroform solution was collected by filtration, concentrated to 1-2 ml by an evaporator, and 10 ml of hexane was added to the concentrated solution to precipitate hexane insolubles. A precipitate was obtained with the AC16 (ORF23) strain, but only a trace of the precipitate was obtained with the CHA1 (ORF23) strain. No precipitate was obtained with the AC16 (CT) strain. AC16 (ORF2
3) After drying the hexane precipitate obtained from the strain, 400M
Hz proton NMR analysis (JEOL, JNM-EX40
0) was performed. FIG. 8 shows an NMR chart. Also,
The correspondence between each peak and the proton of polyester P (3HB-co-3HH) was indicated by a number. From the analysis, the 3HH fraction was 9.7%. The production amount of the polyester was 0.1% of the yeast dry weight. CHA1 (ORF2
3) Similarly, polyester was confirmed in the strain, but the production amount was 0.05% or less. Thus, it was found that a copolymerized polyester P (3HB-co-3HH) can be produced using the ADE1 gene-disrupted strain of Candida maltosa of the present invention. Furthermore, the transformant of the present disrupted strain was obtained from the ADE1 gene mutant yeast CH obtained by the mutation treatment.
The ADE1 gene-disrupting yeast of the present invention was shown to be more excellent in polyester production ability than A1, and the usefulness of the ADE1 gene disrupted yeast of the present invention was clarified.

[0083]

According to the present invention, the usefulness of a strain in which the ADE1 gene of yeast has been disrupted has been shown. The ADE1 gene-disrupted strain of Candida maltosa can be expected to be used as a host yeast for gene recombination for gene expression and production of gene expression products. Further, different auxotrophy can be added, which leads to the development of a yeast that can be safely used as a recombinant.

[0084]

[Sequence List] <110> Kaneka Corporation Kaneka Corporation <120> Gene disrupted strain <130> TKS-4360 <160> 13 <210> 1 <211> 1820 <212> DNA <213> Candida maltosa <220 > <221> CDS <222> 538..1413 <223> Ade1 <400> 1 gatccccttc ttcaaacctt taaatgacat tgtttcgttt ctctatgttt ggtatcggtt 60 cttcttcttc ttcaaaaaaa aggggggcac tattcaaaaa aaaatattat aacagtatga 120 tttttttccc tctcccgtcg attgaggttt tttttttctc tttcgtcttg gtcttttgct 180 tttcactcca aaaatggaaa cacgcgcggc tcaactcgaa atccgtgatc aaaaaaataa 240 aggctgtgag tttcgagcca ataattatga attagtggta ttttttttaa agataaataa 300 tcaagaatcg cattagggag acgaatatgc gttattcaaa taaaaagaca attcttttag 360 ggtagcattt cccttcaagt tcatcccaca tgtacattaa tgtcaatgat gtcgcagaag 420 ttaaattagc agaagaaaaa aaaaatgtga attactccga gtcaactctt ctttctcttc 480 ttctttttct tctttatcac cataatcacc accaccacca ccaccaccag ctcccagatg 540 acttcaacta acttagaagg aactttccca ttgattgcca aaggtaaagt cagagatatt 600 taccaagttg acgacaacac tctttt attc gttgctactg atagaatttc cgcatacgat 660 gtgattatgt ctaatggtat cccaaataaa ggtaaaatct taaccaaatt gtctgaattc 720 tggtttgatt tcttgccaat tgaaaaccat ttaatcaaag gagacatttt ccaaaaatat 780 cctcaactag aaccatatag aaaccaattg gaaggcagat ccttacttgt tagaaaattg 840 aaattgatcc ctcttgaagt tattgttaga ggttacatca ccggttccgg ctggaaagaa 900 taccaaaaat ctaaaaccgt ccacggtatt cctattggtg atgtggttga atcacaacaa 960 atcactccta tcttcacccc atccactaaa gcagaacaag gtgaacatga tgaaaatatc 1020 accaaagaac aagctgacaa gattgttgga aaagaattat gtgatagaat tgaaaaaatt 1080 gctattgatt tgtacaccaa agccagagat tacgctgcca ctaaaggaat tattatcgct 1140 gatactaaat ttgaatttgg tttagatggt gacaacatcg ttcttgttga cgaagtttta 1200 actccagatt cttccagatt ctggaatgct gctaaatacg aagttggtaa atctcaagac 1260 tcttacgata aacaattttt gagagattgg ttaacttcta atggtgttgc tggtaaagat 1320 ggtgttgcta tgcctgaaga cattgtcact gaaaccaaga gcaaatacgt tgaagcttac 1380 gaaaatttaa ctggtgacaa atggcaagaa taaattaagg atatctatta ttaaagcttt 1440 ctatttatcc caaactttcg tagtattttc tgacatg ttc agatgttttt actttatctt 1500 tcctgaaatt tttgatttct aaccgactct tgcatgtagc tcttgataat gcaacatatg 1560 cttgaccatt agcaaaactt ctacctaaat ctattttgac tctgtccaaa gtttgacctt 1620 gagctttgtg gatcgacatc gcccacgaca agatcatttg gtttgttttt atggtgggtt 1680 attggcactt ggtgcaactg atggtttaac tttggaagag gctaagaaat tgaagacttg 1740 gaatgaagaa cgtgcatctg atttcaaatt gggtgaagaa ttgacttata cttgttataa 1800 aatgtatcat gatgttgatc 1820 <210> 2 <211> 1032 <212> DNA <213> Candida maltosa <400> 2 attataacag tatgattttt ttccctctcc cgtcgattga ggtttttttt ttctctttcg 60 tcttggtctt ttgcttttca ctccaaaaat ggaaacacgc gcggctcaac tcgaaatccg 120 tgatcaaaaa aataaaggct gtgagtttcg agccaataat tatgaattag tggtattttt 180 tttaaagata aataatcaag aatcgcatta gggagacgaa tatgcgttat tcaaataaaa 240 agacaattct tttagggtag catttccctt caagttcatc ccacatgtac attaatgtca 300 atgatgtcgc agaagttaaa ttagcagaag aaaaaaaaaa tgtgaattac tccgagtcaa 360 ctcttctttc tcttcttctt tttcttcttt atcaccataa tcaccaccac caccaccacc 420 accagctccc agatgacttc aactaactta gaagga actt tcccattgat tgccaaaggt 480 aaagtcagag atatttacca agttgacgac aacactcttt tattcgttgc tactgataga 540 atttccgcat acgatgtgat tatgtctaat ggtatcccaa ataaaggtaa aatcttaacc 600 aaattgtctg aattctggtt tgatttcttg ccaattaact tctaatggtg ttgctggtaa 660 agatggtgtt gctatgcctg aagacattgt cactgaaacc aagagcaaat acgttgaagc 720 ttacgaaaat ttaactggtg acaaatggca agaataaatt aaggatatct attattaaag 780 ctttctattt atcccaaact ttcgtagtat tttctgacat gttcagatgt ttttacttta 840 tctttcctga aatttttgat ttctaaccga ctcttgcatg tagctcttga taatgcaaca 900 tatgcttgac cattagcaaa acttctacct aaatctattt tgactctgtc caaagtttga 960 ccttgagctt tgtggatcga catcgcccac gacaagatca tttggtttgt ttttatggtg 1020 ggttattca t10tt <tt> attr <tt> attr> ctct 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aacaaaccaa atgatcttgt cgtg 24 <210> 5 <211> 264 <212> DNA <213> Candida maltosa <220> <221> CDS <223> probe <400> 5 aacttctaat gg tgttgctg gtaaagatgg tgttgctatg cctgaagaca ttgtcactga 60 aaccaagagc aaatacgttg aagcttacga aaatttaact ggtgacaaat ggcaagaata 120 aattaaggat atctattatt aaagctttct atttatccca aactttcgta gtattttctg 180 acatgttcag atgtttttac tttatctttc ctgaaatttt tgatttctaa ccgactcttg 240 catgtagctc ttgataatgc aaca 264 <210> 6 <211> 1785 <212> DNA <213> Artificial Sequence <220 > <221> CDS <222> 1..1785 <223> ORF2 <400> 6 atg tct caa cca tct tat ggt cca ttg ttc gaa gct ttg gct cat tac 48 aat gat aaa ttg ttg gct atg gct aaa gct caa acc gaa aga act gct 96 caa gcc ttg ttg caa act aac ttg gat gat ttg ggt caa gtt ttg gaa 144 caa ggt tct caa caa cca tgg caa ttg att caa gct caa atg aat tgg 192 tgg caa gat caa tta aaa ctt atg cag tta aaa tct gct 240 ggt caa cca tct gaa cca gtt att act cca gaa aga tct gat aga aga 288 ttt aaa gct gaa gct tgg tct gaa caa cca att tat gat tac tta aaa 336 caa tcc tat ttg tt cat tt gg ga cat gct tct gtt gat gct 384 ttg gaa ggt gtc cca caa aaa tct aga gaa aga ttg aga ttc t tt act 432 aga caa tac gtc aac gct atg gct cca tct aat ttc ttg gct act aac 480 cca gaa ttg tta aaa ttg act ttg gaa tcc gat ggt caa aat ttg gtt 528 aga ggt ttg gct tta ttg gtt ga gct gat caa 576 tta aac att aga ttg act gat gaa tcc gct ttt gaa tta ggt aga gat 624 ttg gct ttg act cca ggt aga gtt gtt caa aga act gaa tta tat gaa 672 tta att caa tac tct cca act gat acc aaa acc cca gtt 720 ttg atc gtt cca cca ttc att aat aaa tat tac att atg gat atg aga 768 cca caa aac tcc ttg gtc gct tgg ttg gtc gct caa ggt caa acc gtt 816 ttc atg att tcc tgg gt aacca caa gct caa att gat 864 tta gat gat tat gtt gtt gat ggt gtc att gct gct ttg gat ggt gtt 912 gaa gcc gct act ggt gaa aga gaa gtt cac ggt att ggt tac tgt att 960 ggt ggt acc gct ttg tct gt tgg ttg gcc gcc aga aga 1008 caa aaa caa aga gtt aga act gct act ttg ttt act act ttg ttg gat 1056 ttc tcc caa cca ggt gaa ttg ggt att ttt att cat gaa cca att atc 1104 gcc gcc tta gaa gcc ca gaa gcca t aaa ggt att atg gat ggt aga 1152 caa ttg gcc gtc tcc ttc tct ttg ttg aga gaa aac tct tta tat tgg 1200 aat tac tat att gat tct tac tta aaa ggt caa tct cca gtt gct ttt 1248 gat ttg ttgtt gat tct act aat gtt gcc ggt aaa act 1296 cat aac tct ttg ttg aga aga tta tat ttg gaa aat caa ttg gtt aaa 1344 ggt gaa tta aaa aaa att aga aac act aga att gat tta ggt aaa gtt aaa 1392 act cca gtt tt tct gcc gtt gat gat cac att gct tta tgg 1440 caa ggt acc tgg caa ggt atg aaa ttg ttc ggt ggt gaa caa aga ttt 1488 tta ttg gcc gaa tcc ggt cat att gct ggt att att aat cca cca gct aaa gac agt ttc tgg cac aat ggt gct gaa gct gaa tct cca 1584 gaa tct tgg ttg gct ggt gcc acc cat caa ggt ggt tcc tgg tgg cca 1632 gaa atg atg ggt ttt att caa aac aga gat gaa ggt tct gaa cca gtc gaa cca gcc 1 cca gaa gaa ggt ttg gct cca gct cca ggt cac tat 1728 gtc aaa gtt aga tta aac cca gtt ttc gct tgt cca acc gaa gaa gat 1776 gct gct taa 1785 <210> 7 <211> 405 <212> DNA <213> Artificial Sequenc e <220> <221> CDS <222> 1..404 <223> ORF3 <400> 7 atg tct gct caa tcc ttg gaa gtt ggt caa aaa gct aga tta tct aaa 48 aga ttc ggt gcc gcc gaa gtt gct gct ttt gct gcc tta tct gaa gat 96 ttc aac cca ttg cac ttg gat cca gct ttt gct gct act acc gcc ttc 144 gaa aga cca atc gtc cat ggt atg ttg tta gct tct tta ttt tcc ggt 192 ttg ttg ggt caa gt ggt tct att tat ttg ggt caa 240 tct tta tct ttc aaa ttg cca gtc ttt gtc ggt gat gaa gtt acc gct 288 gaa gtt gaa gtt act gct ttg aga gaa gat aaa cca att gct act ttg g 336 act act aga att ggt gct tta gct gtt acc ggt gaa 384 gct gtt gtc aaa ttg cca taa 405 <210> 8 <211> 1017 <212> DNA <213> Candida maltosa <220> <223> promoter ALK1p <400> 8 atgcatgaac aggatttaat cccaagaaaa aagtctattt tctattttca caaggaaact 60 ggaaaaacct ttttgtgttt tgaagtagct ccgtaataac ctgtaaaaaa ataaattttg 120 aagatttgac ttgctgatga aaatgctatc agtgtagctc tagacttgat actagactat 180 gatggcaaca catggtggtc aacgtaccacaagagagagagagagagagagagagagagagagagagagagagagaccag atcagagcaagagagagagagagagagagagagagagagagagagagagagagagagagagagagatag ggacaaaaga aaaactcgag agaaaaagtc aaattggtgt aaaattggct 300 atttttggta ctttcctaat ggggaaatta attgtttaaa attccagttt ttccagagtt 360 aagatttcga ccaattattt ttaatccata tgatcttcat cattatcaac ttgtgaaaaa 420 taataatcga ggtacgttta atacgagata ttagtctacg gctatgaatg ttggatatac 480 ttcattgacg atcagaagct tgattggtta ttcaggtgca tgtgtggata taaacccaac 540 aaattatcta gcaactgtgc cttccccaca ttggtcaaag aaaccctaaa gcaaattaaa 600 atctggataa ataaatcatt catttcacat tttccggtta gtataaggtt ttttaaattt 660 ttttttacag tttagccctt tcaattacca aatacggtaa caatgtgctt tgtaacatgc 720 aggggatttt ctccgttgct gttttctcca catgctttta atgtgtaata aattaaaaaa 780 attacaaaga aaaaccggca tataagcatc ggagtttaca ttgttaacta actgcaaaat 840 ggcgatgttt caaatcaaca aaatttaaaa aaaccccaaa aaaaaagtat catataaatt 900 aaactcaaaa tccttttgat tgcataaaat ttttaaatct cttctttttt ttctttttta 960 ctttcttatc tattctattc tttttttata tatctaattc atttataaca tctggtc 1017 <210> 9 <211> 218 <212 > DNA <213> Candida maltosa <220> <223> terminater ALK1t <400> 9 atagatggat ttttc ttttt tatgtgtatt tccggttaat aaatgtttaa attttttttt 60 taataaaaat atttgtagtt atttatatgc aaaaaaaaaa aatattcaaa gcaatcttcc 120 tttctttctt tatctttccc ccatgctaag gtctaaaaca ccacaactta aaacccaact 180 taaccgtata atctaat <a> 220> ca> 400> 10 tttttcagct ggagctcgtc gacatgcatg aacaggattt aatccc 46 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ccggaattcc atatgcagat gttataaatg aattagata 39 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cggaagctta tagatggatt tttctttttt at 32 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 13 tttttgatatc gagctcgtcg acatgcatct ttggagattg atctt 45

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Simple diagram of pUC119-ADE1 and AD.
A method for preparing E1 disruption DNA is shown.

FIG. 2 shows an expression cassette pUTA-ORF23 for expressing a polyester synthase and an enoyl-CoA hydratase derived from Aeromonas caviae (top of FIG. 2) used in the present invention, and a control cassette pUTA1 containing no genes expressing both enzymes (bottom of FIG. 2). ).

FIG. 3 shows one of ADE1 gene-disrupted strains by PCR.
The results of the following screening are shown. C16 and C1
7 were considered disrupted.

FIG. 4 shows confirmation of ADE1 gene disruption by genomic Southern hybridization. C16
And C17 were normally destroyed.

FIG. 5 shows a restriction map of the ADE1 gene disrupted Candida maltosa chromosome in the vicinity of the disrupted ADE1 gene, as revealed by Southern hybridization.

FIG. 6 shows a growth curve of ADE1 gene-disrupted strain AC16 in YPD medium.

FIG. 7 shows a growth curve of ADE1 gene-disrupted strain AC16 when coconut oil, palm oil, and tetradecane were used as carbon sources.

FIG. 8 shows a 400 MHz proton NMR chart of polyester P (3HB-co-3HH) produced by AC16 (ORF23) strain. The assignment of each proton of the polyester is indicated by a number.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12P 7/62 C12R 1:72) C12R 1:72) C12N 15/00 ZNAA (72) Inventor Masamichi Takagi 1-31-10 Sakaemachi, Fuchu-shi, Tokyo (72) Inventor Akinori Ota 57-2 Brother, Omiya-shi, Saitama F-term (reference) 4B024 AA17 AA20 BA07 BA80 DA12 GA11 HA20 4B064 AD83 CA06 CA19 CB24 CC24 DA20 4B065 AA10Y AA73X AB01 AC20 BA02 CA12 CA54

Claims (12)

[Claims] 1. A yeast in which the ADE1 gene of chromosomal DNA has been disrupted by homologous recombination with an ADE1 DNA fragment. 2. The method according to claim 1, wherein the yeast is of the genus Acycloconidium, the genus Ambrosiozyma, the genus Arthroascus, the genus Arxiozyma, the genus Ashvia, the genus Babjevia, the genus Bensingtonia, the genus Botryoascus, the genus Botryozaiima, the genus Brettanomies, burella. Genus, Burellomyces, Candida, Cisteromyces, Clavispora,
Cryptococcus, Cystophilus genus,
Genus Debaryomyces, genus Deccala, genus Dipodascopsis, genus Dipodascus, Eniera, genus Endomychosera, elemascus, elemosesium, erythrobasidium, genus Ferromyces, philobasidium, galactomyces, geotricum Genus, genus Geirermondera, genus Hanseniaspora, genus Hansenula, genus Hasegawaae, genus Holtermannia, genus Hormoascus,
Genus Hyfopichia, Isathenkia, Chloekera, Chloekeraspora, Kluyveromyces, Condoa, Chryssia, Kurzmanomyces, Leucosporidium, Lipomyces, Roderomyces, Malassezia, Methshnikowia , Murakia, Mikesozaima, Nadsonia, Nakazawaea, Nematospora, Ogataea, Auspolidium, Pachisolen, Faticospora, Phaffia, Pichia, Rhodosporium, Rhodotorula,
Saccharomyces, Saccharomycordes, Saccharomycopsis, Cytoela, Sakagutia, Satanospora, Schizoblastosporion, Schizosaccharomyces, Schwaniomyces, Sporidiobolas, Su Genus Poloboromyces, genus Sporopachydemia, genus Stefanoascus, genus Sterigmatomyces, genus Sterigmatosporidium, genus Symbiotaflina, genus Sympodiomyces, genus Sympodiomycopsis, genus Torricola, Genus Trichosporon, Genus Trigonopsis, Genus Tutsyaea, Genus Udeniomyces, Genus Waltmyces, Genus Wikerhamia, Genus Wikerhamiera, Genus Willioptis, Genus Yamadazaima, Genus Yarrowia, Genus Zygoascus, Genus Zygosaccharomyces, Genus Zygowili Pushisu genus, or Zaigozaima genus ADE1 gene disrupted yeast according to claim 1, wherein either. 3. The ADE1 gene-disrupted yeast according to claim 1, wherein the yeast is any one of the genus Candida, the genus Yarrowia, the genus Kluyveromyces, and the genus Hansenia spora. 4. The ADE1 gene disrupted yeast according to claim 1, wherein the yeast is of the genus Candida. 5. The ADE1 gene-disrupted yeast according to claim 4, wherein the yeast is Candida maltosa. 6. The method according to claim 6, wherein the yeast is Candida maltosa AC16.
(FERM BP-7366).
DE1 gene disrupted yeast. 7. The yeast ADE1 gene-disrupted yeast transformant according to claim 1, which is transformed with a DNA sequence containing a homologous or heterologous gene. 8. The transformant according to claim 7, wherein the ADE1 gene-disrupted yeast belongs to the genus Candida. 9. The ADE1 gene-disrupted yeast is Candida.
The transformant according to claim 8, which is maltosa. 10. The transformant according to claim 9, wherein the ADE1 gene disrupted yeast is Candida maltosa AC16 (FERM BP-7366). 11. A gene expression product, wherein a homologous or heterologous gene expression product is collected from a culture obtained by culturing the transformant according to any one of claims 7 to 10. Manufacturing method. 12. A method for producing a polyester, wherein the gene expression product according to claim 11 is a polyester.